Nuclear envelopes of eukaryotic cells provide a means of spatially separating transcription and translation and allow for extensive processing of RNAs before their use in the cytoplasm. Selective and regulated transport of macromolecules through nuclear pore complexes enables cells to control the delivery of molecules to the cytoplasm and to monitor the maturation and functionality of these cargos (Johnson et al., Trends Biochem. Sci., 2002, 27, 580-585).
Exportin 5, also known as XPO5, belongs to a large family of karyopherins, which are proteins that mediate the transport of cargo between the nuclear and cytoplasmic compartments in a RanGTP-dependent manner. First cloned in 1999 from an adult brain cDNA library, exportin 5 shares significant homology with the general nucleoporin export receptor CRM1 (Brownawell et al., J. Cell. Biol., 2002, 156, 53-64).
RT-PCR studies have detected low to moderate expression in heart, brain, lung, liver, skeletal muscle, kidney, pancreas, spleen, testis, and ovary. The deduced 1,205-amino acid protein has a calculated molecular mass of about 136 kD and exportin 5 homologues have been cloned in Xenopus and Arabidopsi (Bohnsack et al., Rna, 2004, 10, 185-191; Bollman et al., Development, 2003, 130, 1493-1504).
To date, a number of different cargo have been identified for exportin 5, including interleukin enhancer binding protein and the Fas-associated death domain protein (Brownawell et al., J. Cell. Biol., 2002, 156, 53-64; Screaton et al., Proc Natl Acad Sci USA, 2003, 100, 5211-5216). Export of interleukin enhancer binding factor 3 (ILF3) was demonstrated by Bromawell et al. (Brownawell et al., J. Cell. Biol., 2002, 156, 53-64). Exportin 5 was shown to bind ILF3 at its double-stranded RNA-binding domain (dsRBD), and this binding is disrupted in the presence of dsRNA (Brownawell and Macara, J. Cell. Biol., 2002, 156, 53-64). Exportin 5 also binds the dsRBD of the spermatid perinuclear binding protein, staufen, and protein kinase R. It is thought that exportin 5 regulates the translocation of these dsRBD proteins to the cytoplasm, where they interact with target mRNAs.
Bohnsack (Bohnsack et al., Embo. J., 2002, 21, 6205-6215) and Calado (Calado et al., Embo. J., 2002, 21, 6216-6224) report that exportin 5 transports the translation elongation factor 1A (eEF1A), and that this transport depends on interaction via aminoacylated tRNAs, thereby ensuring the confinement of translation to the cytoplasm.
By microinjection in Xenopus oocyte, Gwizdek et al. (Gwizdek et al., J. Biol. Chem., 2003, 278, 5505-5508) found that exportin 5 mediated the nuclear export of adenovirus VA1 RNA. The authors demonstrated that exportin 5 preferentially recognizes and transports RNAs containing a minihelix motif, which is a double-stranded stem structure. Gwizdek et al. (Gwizdek et al., J. Biol. Chem., 2004, 279, 884-891) further demonstrated that the formation of the exportin 5 ILF3 complex is facilitated by the simultaneous binding of exportin 5 to minihelix-containing RNAs.
Exportin 5 has also been shown to mediate the efficient nuclear export of short microRNA (miRNA) precursors (pre-miRNAs) in both Xenopus and human cell lines. (Bohnsack et al., Rna, 2004, 10, 185-191; Lund et al., Science, 2004, 303, 95-98; Yi et al., Genes Dev., 2003, 17, 3011-3016). Pre-miRNAs are endogenous, approximately 70 nucleotide-long hairpin (also known as stem-loop or foldback) precursor transcripts that can form local hairpin structures. Once in the cytoplasm, these miRNA hairpin precursors are processed such that a single-stranded mature miRNA molecule is generated from one arm of the hairpin precursor. Alternatively, a polycistronic miRNA precursor transcript may contain multiple hairpins, each processed into a different, single mature miRNA. The current model is that either the primary miRNA transcript or the hairpin precursor is transported out of the nucleus by and cleaved by Dicer to yield a double-stranded intermediate. Only one strand of this short-lived intermediate accumulates as the mature miRNA (Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, Plant Physiology, 2003, 132, 709-717; Shi, Trends in Genetics, 2003, 19, 9-12). Mature miRNAs are approximately 22 nucleotide-long RNAs that can play important regulatory roles by targeting mRNA for cleavage or translational repression. Lund et al. (Lund et al., Science, 2004, 303, 95-98) further demonstrated that exportin 5 binds correctly processed pre-miRNAs directly and specifically, but interacts only weakly with extended pre-miRNAs that yield incorrect miRNAs when processed. This observation suggests that exportin 5 is key to miRNA biogenesis and may help coordinate nuclear and cytoplasmic processing steps.
Antisense technology is an effective means of reducing the expression of specific gene products and therefore is uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of exportin 5 expression. The present invention provides compositions and methods for modulating exportin 5 expression.